Preparation of hymenialdisine derivatives and use thereof

ABSTRACT

The synthesis and biological activity of indoloazepines and acid amine salts thereof which are structurally related to naturally-occurring hymenialdisine is disclosed. Naturally-occurring hymenialdisine obtained from the sponge is a potent inhibitor of production of cytokines interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). The chemically-synthesized indoloazepines of the invention also inhibit production of IL-2 and TNF-α. The indoloazepines are useful for treating inflammatory diseases, particularly diseases associated with kinases NF-κB or GSK-3β activation or NF-κB activated gene expression products. The indoloazepines are useful for the treatment of cancer by the inhibition of kinases CHK1 and CHK2.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Provisional Application Ser.No. 60/471,671, filed May 19, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

[0003] Reference to a “Computer Listing Appendix submitted on Disc”

[0004] Enclosed is Computer Listing Appendix submitted on disc.

BACKGROUND OF THE INVENTION

[0005] (1) Field of the Invention

[0006] The present invention related to the synthesis and biologicalactivity of indoloazepines and acid amine salts thereof which arestructurally related to naturally-occurring hymenialdisine. Thechemically-synthesized indoloazepines inhibit production of IL-2 andTNF-α. Exposure of the chemically-synthesized indoloazepine to mammalianJurkat leukemia T-cells and THP-1 cells results in a dose responseinhibition of IL-2 production and TNF-α production, respectively. Theindoloazepines are useful for treating inflammatory diseases,particularly diseases associated with NF-κB or GSK-3β activation andNF-κB activated gene expression.

[0007] (2) Description of Related Art

[0008] Elevated levels of cytokines, such as interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), andtumor necrosis factor-α (TNF-α) have been linked to many inflammatorydisorders including Crohn's disease and psoriasis, and play an essentialrole in the pathogenesis of rheumatoid arthritis and osteoarthritis(Gerard et al., Nat. Immunol. 2: 108-115 (2001); Inoue et al., Inflamm.Res. 50: 65-72 (2001); Polisson, Curr. Rheumatol. Rep. 3: 489-495(2001); Miossec, Cell Mol. Biol. (Noisy-le-grand) 47: 675-678 (2001);Roshak et al., J. Pharmacol. Exp. Ther. 283: 955-961 (1997); Harris, N.Engl. J. Med. 322: 1277-1289 (1990)).

[0009] Using anti-inflammatory drugs to inhibit cytokines, in particularTNF-α, has been successful in several clinical trials for treatingrheumatoid arthritis (Moreland, J. Rheumatol. 26 Suppl. 57: 7-15 1999);Moreland, et al., N. Engl. J. Med. 337: 141-147 (1997)). However, thereis variability in responses to these anti-inflammatory drugs because ofthe complex network of alternative cytokine mediated pathways (Miossec,Cell Mol. Biol. (Noisy-le-grand) 47: 675-678 (2001); Handel et al.,Clin. Exp. Pharmacol. Physiol. 27: 139-144 (2000)). Using drugs whichinhibit transcription factors that control the expression of severalpro-inflammatory mediators, such as the nuclear transcription factorNF-κB, may overcome response variability and may provide an alternativestrategy for treating a wide variety of inflammatory disorders (Makarov,Arthritis Res. 3: 200-206 (2001); Tak, J. Clin. Invest. 107: 7-11(2001); Roshak et al., Curr. Opin. Pharmacol. 2: 316-321 (2002);Yamamoto and Gaynor, J. Clin. Invest. 107: 135-142 (2001); Feldmann etal., Ann. Rheum. Dis. 61 Suppl. 2: ii13-18 (2002); Barnes and Karin, N.Engl. J. Med. 336: 1066-1071 (1997); Lee and Burckart, J. Clin.Pharmacol. 38: 981-993 (1998); Baldwin, Ann. Rev. Immunol. 14: 649-683(1996); Miagkov et al., Proc. Natl. Acad. Sci. USA 95: 13859-13864(1998); Guttridge et al., Mol. Cell Biol. 19: 5785-5799 (1999); Baeuerleand Henkel, Ann. Rev. Immunol. 12: 141-179 (1994)).

[0010] Because of its critical role in the regulation of inflammatoryresponses, NF-κB has become an increasingly significant therapeutictarget for controlling diseases such as asthma, rheumatoid arthritis,multiple sclerosis, and Alzheimer's disease (Tak, J. Clin. Invest. 107:7-11 (2001); Yamamoto and Gaynor, J. Clin. Invest. 107: 135-142 (2001);Barnes and Karin, N. Engl. J. Med. 336: 1066-1071 (1997); Boland,Biochem. Soc. Trans. 29: 674-678 (2001); Hart et al., Am. J. Respir.Crit. Care Med. 158: 1585-1592 (1998); Yamamoto and Gaynor, Curr. Mol.Med. 1: 287-296 (2001)).

[0011] Hymenialdisine is a bromopyrrole alkaloid which was originallyisolated from the marine sponges Axinella verrucosa and Acantellaaurantiaca. Its structure was established on the basis of X-raycrystallography (Cimino et al., Tet. Lett. 23: 767-768 (1982)). Thestructure of hymenialdisine is shown in FIG. 1A. Hymenialdisine has beenfound to inhibit various proinflammatory cytokines, such as IL-1, IL-6,IL-8, and nitric oxide in a variety of cell lines (Inoue et al.,Inflamm. Res. 50: 65-72 (2001); Roshak et al., J. Pharmacol. Exp. Ther.283: 955-961 (1997); Breton and Chabot-Fletcher, J. Pharmacol. Exp.Ther. 282: 459-466 (1997); Badger et al., J. Pharmacol. Exp. Ther. 290:587-593 (1999)). Investigation of the promising anti-inflammatoryproperties of hymenialdisine revealed that it inhibits cytokineproduction by inhibiting the NF-κB signaling pathway (Roshak et al., J.Pharmacol. Exp. Ther. 283: 955-961 (1997); Breton and Chabot-Fletcher,J. Pharmacol. Exp. Ther. 282: 459-466 (1997); Badger et al., J.Pharmacol. Exp. Ther. 290: 587-593 (1999); Badger et al., OsteoarthritisCartilage 8: 434-443 (2000)). Gel shift analysis showed that thisinhibition was that hymenialdisine selectively reduced NF-κB nuclearbinding and not the binding of other transcription factors such asC/EBP, AP-1, or SP1 (Roshak et al., J. Pharmacol. Exp. Ther. 283:955-961 (1997)).

[0012] Recently, Meijer et al. reported that the potent NF-κB inhibitorhymenialdisine acts as a competitive nanomolar inhibitor of thecyclin-dependent kinases GSK-3β and CK1 (Meijer et al., Chem. Biol. 7:51-63 (2000)). Crystallographic data showed that hymenialdisine binds tothe ATP binding pocket of the GSK-3β and CK1 kinases (Meijer et al.,Chem. Biol. 7: 51-63 (2000)). Considering the potential relationshipbetween NF-κB activation and GSK-3, that might suggest a potentialpathway for this kinase inhibitor (Meijer et al., Chem. Biol. 7: 51-63(2000); Ali et al., Chem. Rev. 101: 2527-2540 (2001); Schwabe andBrenner, Am. J. Physiol. Gastrointest. Liver Physiol. 283: G204-211(2002)). In addition, Ireland et al. identified hymenialdisine as a verypotent MEK-1 inhibitor with low nanomolar IC₅₀ values. This suggeststhat hymenialdisine may be useful as an antiproliferative agent(Tasdemir et al., J. Med. Chem. 45: 529-532 (2002)).

[0013] Hymenialdisine and various derivatives thereof such asdebromohymenialdisine have been disclosed in the following patents andpublished patent applications.

[0014] U.S. Pat. No. 5,565,448 to Nambi et al. discloses medicants whichcontain hymenialdisine or debromohymenialdisine and which are used toinhibit protein kinase C and U.S. Pat. No. 5,616,577 to Nambi et al.discloses methods using hymenialdisine or debromohymenialdisine toinhibit protein kinase C.

[0015] U.S. Pat. No. 5,591,740 to Chipman et al. discloses usingcompositions comprising hymenialdisine or debromohymenialdisine to treatosteoarthritis.

[0016] U.S. Pat. No. 5,621,099 to Annoura et al. discloses a method forsynthesizing hymenialdisine, bromohymenialdisine, and related compounds.

[0017] U.S. Pat. Nos. 5,834,609, 6,103,899, and 6,218,549, all to Horneet al. disclose bicyclic aminoimidizole compounds which have anti-tumorand antimicrobial activity.

[0018] U.S. Pat. No. 6,197,954 B1, 6,211,361, 6,528,646, and publishedU.S. Patent Application No. 2001/0012891A1, all to Horne et al.,disclose processes for synthesizing hymenialdisine, related compounds,and their intermediates.

[0019] Published U.S. Patent Application No. 20030060457A1 to Schafferet al. discloses that hymenialdisine is a cdk inhibitor which can beused as an inhibitor of gene expression, replication, and reactivationin pathogenic agents.

[0020] EP1106180A1 and WO0141768A2 to Meijer disclose usinghymenialdisine and related compounds such as debromohymenialdisine toinhibit cyclin dependent kinases, GSK-3β, and casein kinase 1 forpreventing and treating neurodegenerative disorders such as Alzheimer'sdisease, diabetes, inflammatory pathologies, and cancers.

[0021] DNA replication is a process that requires great accuracy andrelies on surveillance mechanisms, which monitor DNA damage and initiateDNA repair (Zhou, B.-B. S., et al., Nature 408 433-439 (2000)). Theinability to carry out DNA repair often leads to the transformation ofnormal cells into malignancies (Martin, N. M. B., J. Photochem.Photobiol. B 63 162-170 (2001)). Upon DNA damage, cell cycle checkpointsget activated, which delay cell cycle progression and allow DNA repair.A multi-faceted involvement of these checkpoint pathways regulates DNArepair, (Zhou, B.-B. S., et al., Nature 408 433-439 (2000; Martin, N. M.B., J. Photochem. Photobiol. B 63 162-170 (2001); and Zhao, S., et al.,Nature 405, 473 (2000)) telomere length (Naito, T., et al., Nat. Genet.20 203-206 (1998); Ritchie, K. B., et al., Mol. Cell Biol. 10 6065-6075(1999)) and the induction of apoptotic cell death (Zhou, B.-B. S., etal., Nature 408 433-439 (2000); and Lowe, S. W., et al., Nature 362847-849 (1993)). Protein kinases regulate a host of cellular processessuch as growth and differentiation, cell proliferation and apoptosis(Sielecki, T. M., et al., J. Med. Chem. 43 1-18 (2000); Sridhar, R., etal., Pharm. Res. 17 1345-1353 (2000); Traxler, P., et al., J. Med. Res.Rev. 21 499-512 (2001); Scapin, G., Drug Discovery Today 7. (2001);Toogood, P. L., Med. Res. Rev. 21 487-498 ((2001); and Bridges, A. J.,Chem. Rev. 101 2541-2572 (2001)). DNA damage caused by radiation orchemotherapy triggers the DNA damage-responsive protein kinases ATM andATR, which activate Chk1 and Chk2. Chk1 and Chk2 in turn phosphorylateCdc25 and prevent Cdc2 activation, resulting in cell cycle arrest(Curman, D., et al., J. Biol. Chem. 276 17914-17919 (2001)). Hence,small molecules that can inhibit the checkpoints may enhance theefficacy of DNA damaging chemotherapeutics or radiation therapy (Rundle,N. T., et al., J. Biol. Chem. 276 48231-48236 (2001); Jackson, J. R., etal., Cancer Res. 60 566-572 (2000); Koniaras, K., et al., Oncogene 207453-7463 (2001); Zhou, B.-B. S., et al., Cancer Biol. Ther. 2 S16-S22(2003); Yu, Q., et al., Cancer Res. 62 5743-5748 (2002)).

[0022] In light of the prior art, there remains a need for other smallmolecules that have activities which exhibit a similar or betterpharmacological profile to hymenialdisine and which are simple andinexpensive to prepare.

SUMMARY OF THE INVENTION

[0023] The synthesis and biological activity of indoloazepines and acidamine salts thereof which are structurally related tonaturally-occurring hymenialdisine is disclosed. Naturally-occurringhymenialdisine obtained from the sponge is a potent inhibitor ofproduction of interleukin-2 (IL-2) (IC₅₀=2.4 μM) and tumor necrosisfactor-α (TNF-α) (IC₅₀=1.4 PM). The chemically-synthesized indoloazepinealso inhibits production of IL-2 and TNF-α. Exposure of thechemically-synthesized indoloazepine to mammalian Jurkat leukemiaT-cells and THP-1 cells resulted in a dose response inhibition of IL-2production (IC₅₀=3.5 μM) and TNF-α production (IC₅₀=8.2 μM),respectively. The indoloazepines are useful for treating inflammatorydiseases, particularly diseases associated with NF-κB activation.

[0024] The chemically-synthesized indoloazepine also inhibits the kinaseCHK2 (IC₅₀=8 μM. The indolozaepines are useful for treating cancer.

[0025] Therefore, the present invention provides a compound of theformula

[0026] and acid amine salts thereof, wherein R₁, R₂, and R₃ are moietiessuch that the compound inhibits kinases or NF-κB or NF-κB mediated geneproducts. In a further embodiment, R₁, R₂, and R₃ are moieties such thatthe compound inhibits a cyclin kinase, particularly wherein the cyclinkinase is GSK-3β or CDK-1. In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound are inhibitors of checkpoint kinases,particularly wherein the kinase is CHK2. In a further embodiment, R₁,R₂, and R₃ are each selected from the group consisting of hydrogen,methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl,hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

[0027] The present invention further provides a compound of the formula

[0028] and acid amine salts thereof, wherein R₁, R₂, and R₃ arenon-reactive groups. In a preferred embodiment, R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a particularly preferred embodiment, R₁, R₂, and R₃are each hydrogen.

[0029] The present invention further provides a compound of the formula

[0030] and acid amine salts thereof, wherein R₁, R₂, and R₃ arenon-reactive groups. In a preferred embodiment, R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a particularly preferred embodiment, R₁, R₂, and R₃are each hydrogen.

[0031] The present invention further provides a compound of the formula

[0032] and acid amine salts thereof, wherein R₁, R₂, and R₃ arenon-reactive groups. In a preferred embodiment, R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a particularly preferred embodiment, R₁, R₂, and R₃are each hydrogen.

[0033] The present invention further provides a process for thepreparation of a compound (IV) of the formula

[0034] herein R₁, R₂, and R₃ are which are non-reactive, which comprises(a) reacting a first compound (I) of the formula

[0035] with methyl sulfonic acid and phosphorus pentoxide at elevatedtemperatures to form a second compound (II) of the formula

[0036] (b) reacting compound (II) with phenyl oxazolone and a transitionmetal catalyst in a solvent to form compound (III) of the formula

[0037] and (c) reacting compound (III) with thiourea and a base in asolvent to form the compound (IV). In a further embodiment of theprocess, in step (b) the transition metal catalyst is titanium chlorideand the solvent is tetrahydrofuran. In a further embodiment of theprocess, in step (c) the base is lithium hydride and the solvent isethanol. In a further still embodiment of the process, the compound (I)is formed by reacting a compound (V)

[0038] with an aqueous base solution, preferably wherein the base islithium hydroxide.

[0039] The present invention further provides a method for inhibiting adisease in an animal, preferably a human, associated with a kinase orNF-κB activation which comprises administering a compound of the formula

[0040] and acid amine salts thereof, wherein R₁, R₂, and R₃ are moietiessuch that the compound inhibits the kinase, NF-κB activation or NF-κBmediated gene products, the compound being administered to the animal inan amount sufficient to substantially inhibit the disease. In a furtherembodiment, R₁, R₂, and R₃ are moieties such that the compound inhibitsa cyclin kinase, particularly wherein the cyclin kinase is GSK-3β orCDK-1. In a further embodiment, R₁, R₂, and R₃ are moieties such thatthe compounds are inhibitors of checkpoint kinases, particularly whereinthe kinase is CHK2. In a further embodiment, R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

[0041] In a preferred embodiment of the method, the disease associatedwith NF-κB activation is an inflammatory disorder. In a more preferredembodiment, the disease associated with NF-κB activation is selectedfrom the group consisting of rheumatoid arthritis, inflammatory boweldisease, asthma, dermatosis, autoimmune disease, tissue and organrejection, Alzheimer's disease, stroke, atherosclerosis, restenosis,cancer, viral infections, osteoarthritis, osteoporosis, and AtaxiaTelangiectasia.

[0042] In a preferred embodiment of the method, the disease associatedwith inhibition checkpoint kinases is cancer.

[0043] The present invention further provides a method for inhibitingproduction of cytokines in an animal, preferably a human, whichcomprises administering a compound of the formula

[0044] and acid amine salts thereof, wherein R₁, R₂, and R₃ are moietiessuch that the compound inhibits the cytokines, the compound beingadministered to the animal in an amount sufficient to inhibit productionof the cytokines. In a further embodiment, R₁, R₂, and R₃ are moietiessuch that the compound inhibits a cyclin kinase, particularly whereinthe cyclin kinase is GSK-3β or CDK-1. In a further embodiment, R₁, R₂,and R₃ are moieties such that the compound are inhibitors of checkpointkinases, particularly wherein the kinase is CHK2. In a furtherembodiment, R₁, R₂, and R₃ are each selected from the group consistingof hydrogen, methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl,acyl, hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

[0045] In a preferred embodiment of the method, the cytokines areproduced by activation of the NF-κB pathway. In a more preferredembodiment, the cytokines are selected from the group consisting ofIL-2, IL-6, IL-8, TNF-α, and combinations thereof.

OBJECTS

[0046] It is an object of the present invention to provide smallmolecules and processes for synthesizing which are useful for treatinginflammatory diseases.

[0047] It is a further object of the present invention to provide smallmolecules and processes for synthesizing which are useful for treatingdiseases associated with NF-κB activation.

[0048] it is further an object of the present invention to provide smallmolecules and processes for synthesizing which are useful for treatingdiseases such as cancer.

[0049] These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1A shows the structure of naturally-occurring hymenialdisine1

[0051]FIG. 1B shows the structure for chemically-synthesizedindoloazepine compound 2.

[0052]FIG. 1C shows the structure for chemically-synthesizedindoloazepine compound 3.

[0053]FIG. 2A shows IL-2 production as measured by ELISA with PDTC.

[0054]FIG. 2B shows IL-2 production as measured by ELISA withhymenialdisine 1.

[0055]FIG. 2C shows IL-2 production as measured by ELISA withchemically-synthesized indoloazepine compound 2.

[0056]FIG. 2D shows IL-2 production as measured by ELISA withchemically-synthesized indoloazepine compound 3.

[0057]FIG. 3 shows EMSA assays for NF-κB-DNA binding inhibition bycompounds 1-3. Lane 1: NF-κB consensus oligo (0.16 pmol/λ)+nuclearextract (+PMA/PHA)+Antibody p65; Lane 2: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA); Lane 3: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (—PMA/PHA); Lane 4: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA)+5 μM PDTC; Lane 5: NF-κB consensusoligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)⁺1 μM PDTC; Lane 6: NF-κBconsensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+5 μM compound 1;Lane 7: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+1μM compound 1; Lane 8: NF-κB consensus oligo (0.16 pmol/λ)+nuclearextract (PMA/PHA)+5 μM compound 2; Lane 9: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA)+1 μM compound 2; Lane 10: NF-κBconsensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+5 μM compound 3;Lane 11: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+1μM compound 3.

[0058]FIG. 4 is a cartoon illustrating the role of NF-κB in activatingtranscription of particular genes such as those encoding variouscytokines.

[0059]FIG. 5 is a chart which compares the effect of compounds 1-3 onTNF-α production in THP-1 cells after stimulation with LPS.

[0060]FIG. 6A compares the cytotoxicity of hymenialdisine to compounds 2and 3. Number of cells were normalized on a scale of 100.

[0061]FIG. 6B shows the growth patterns in CEM cells at differentconcentrations of compound 2. Number of cells were normalized on a scaleof 100.

[0062]FIG. 7A is a graph showing inhibition of CDK1 and GSK-3β byhymenialdisine 1. x=CDK1 and ⋄=GSK-3β.

[0063]FIG. 7B is a graph showing inhibition of CDK1 and GSK-3β bycompound 2. x=CDK1 and ⋄=GSK-3β.

[0064]FIG. 7C is a graph showing inhibition of CDK1 and GSK-3β bycompound 3. x=CDK1 and ⋄=GSK-3β.

[0065]FIG. 8 is a table showing inhibition of the kinases: CK1(h),CK2(h), MEK1(h), PKCa(h), PKCbII(h), CHK1 and CHK2.

[0066]FIG. 9 is a graph showing inhibition of CHK1 and CHK2 by compound2.

DETAILED DESCRIPTION OF THE INVENTION

[0067] All patents, patent applications, government publications,government regulations, and literature references cited in thisspecification are hereby incorporated herein by reference in theirentirety. In case of conflict, the present description, includingdefinitions, will control.

[0068] The present invention includes all hydrates, solvates, complexes,and prodrugs of the compounds of this invention. Prodrugs are anycovalently bonded compounds which in vivo releases the active parentdrug having the formula The present invention further includes compoundsand prodrugs of such compounds which have the formula

[0069] and acid amine salts thereof, wherein R₁, R₂, and R₃ are moietiessuch that the compound inhibits NF-κB in human Jurkat Leukemia T-cellsactivated to transcribe NF-κB mediated gene transcription by phorbolmyristate acetate (PMA). In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase, particularlywherein the cyclin kinase is GSK-3β or CDK-1. In a further embodiment,R₁, R₂, and R₃ are each independently selected from the group consistingof methyl, alkyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cyclic, heterocyclic, substituted cyclic, andcombinations thereof; and, the R₁ and R₂ can be interconnected.Preferably, the R₁ and R₂ are interconnected members of an aryl orcyclic ring which can be substituted or comprise heteroatoms or both.Preferably, R₃ is hydrogen. The term “halo” includes F, Cl, Br, and I.The alkyl, aryl, acyl, cyclo includes substituted and heteroatom speciesas well as the unsubstituted species. If a chiral center or another formof an isomeric center is present in a compound of the present invention,all forms of such isomer or isomers, including enantiomers anddiastereomers, are intended to be covered herein. Compounds containing achiral center may be used as a racemic mixture, an enantiomericallyenriched mixture, or the racemic mixture may be separated usingwell-known techniques and an individual enantiomer may be used alone. Incases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention. In cases wherein compounds may exist in tautomeric forms,such as keto-enol tautomers, each tautomeric form is contemplated asbeing included within this invention whether existing in equilibrium orpredominantly in one form.

[0070] The compounds disclosed herein are provided in organic solventssuch as DMSO or other organic solvents which are pharmaceuticallyacceptable. The compounds disclosed herein are also provided as theiramine acid salts. For example, the compounds herein are prepared in anappropriate solvent as taught herein. An excess of an acid such asacetic, hydrochloric, hydrobromic, hydrofluoric, maleic, methansulfonic,phosphoric, succinic, sulfuric, trifluoroacetic, or sulfuric acid isadded which produces the compound as its acid salt. The acid salts ofthe compounds are more soluble in water and other pharmaceuticallyacceptable aqueous solutions.

[0071] In light of the need for small molecule inhibitors of NF-κBmediated gene transcription, the present invention provides chemicalsynthesis and methods of use for the compounds of the above formula. Ina preferred embodiment, the compounds have the formula

[0072] and acid amine salts thereof, wherein R₁₁ R₂′ and R₃ are moietiessuch that the compound inhibits NF-κB in human Jurkat Leukemia T-cellsactivated to transcribe NF-κB mediated gene transcription by phorbolmyristate acetate (PMA). In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase, particularlywherein the cyclin kinase is GSK-3β or CDK-1. In a further embodiment,R₁, R₂, and R₃ are each independently selected from the group consistingof hydrogen, methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl,acyl, hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

[0073] In light of the need for small molecule inhibitors of checkpointkinases, the present invention provides chemical synthesis and methodsof use for the compounds of the above formula. In a preferredembodiment, the compounds have the formula

[0074] and acid amine salts thereof, wherein R₁, R₂, and R₃ are moietiessuch that the compound inhibits checkpoint kinases such as CHK1 andCHK2. In a further embodiment, R₁, R₂, and R₃ are each independentlyselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

[0075] In a preferred embodiment, the present invention provides theindoloazepine, compound 2, which has the formula

[0076] and acid amine salts thereof. A comparison of compound 2 tohymenialdisine 1 and compound 3, a methylated variant of compound 2, isshown in FIGS. 1A, 1B, and ° C.

[0077] Hymenialdisine 1 inhibits a wide variety of proinflammatorycytokines such as IL-2, IL-6, IL-8, and nitric oxides through inhibitionof the NF-κB signaling pathway. Compound 2 also substantially inhibitsNF-κB mediated cytokine production. The examples herein show thatcompound 2, in particular, inhibits IL-2 and TNF-α production with IC₅₀values similar to the IC₅₀ values for the naturally-occurringhymenialdisine 1. Compound 2 and its derivatives, including compoundswith the above formula, substantially inhibits NF-κB mediated cytokineproduction, including IL-2, IL-6, IL-8, and nitric oxides. At theconcentrations tested, compound 3 did not appear to have significantinhibitory activity. Because compounds 2 and 3, and derivatives thereof,including compounds with the above formula, can be chemicallysynthesized, they provide a convenient source of hymenialdisine-relatedsmall molecules which can be used for treating a variety of inflammatorydiseases where it is desirable to inhibit NF-κB-mediated cytokineproduction. The compounds, like hymenialdisine, are also GSK-3βinhibitors (See WO 03/027275 to Hellberg et al.).

[0078] The naturally-occurring hymenialdisine 1 anddebromohymenialdisine inhibit the checkpoint kinases CHK1 and CHK2 atlow micromolar concentrations. Compound 2 is a much more potent andselective inhibitor of inhibited checkpoint kinases than thenaturally-occurring hymenialdisine 1 and debromohymenialdisine. Theexamples herein show that compound 2, in particular, inhibits CHK1 withan IC₅₀ value of 237 nanoMolar concentration and inhibits CHK2 with anIC₅₀ value of 8 nanoMolar concentration. This shows that thesynthetically prepared indoloazepines have an improved overall kinaseprofile and significantly improves its kinase selectivity.

[0079] Therefore, the present invention provides pharmaceuticalcompositions comprising one or more of the compounds with the aboveformula which are inhibitors of transcription factor NF-κB, and methodsfor treating diseases in which activation of NF-κB or activity of GSK-3βis implicated. More specifically, the present invention providescompositions and methods of treatment of a variety of diseasesassociated with NF-κB or GSK-3β activation or GSK-31 inflammatorydisorders; particularly rheumatoid arthritis, inflammatory boweldisease, and asthma; dermatosis, including psoriasis and atopicdermatitis; autoimmune diseases; tissue and organ rejection; Alzheimer'sdisease; stroke; atherosclerosis; restenosis; cancer, includingHodgkin's disease; a variety of viral infections, including AIDS;osteoarthritis; osteoporosis; glaucoma; and, Ataxia Telangiectasia byadministering to an animal or human in need thereof a compoundcomprising the above formula.

[0080] Therefore, the present invention also provides pharmaceuticalcompositions comprising one or more of the compounds with the aboveformula which are inhibitors of checkpoint kinases, and methods fortreating diseases in which checkpoint kinases are implicated. Morespecifically, the present invention provides compositions and methods oftreatment of a variety of diseases associated with checkpoint kinasessuch as cancer.

[0081] Without intending to be bound by any particular theory, theinhibition of NF-κB by the compounds of the above formula is believed tobe exerted in the nucleus of the cell as shown in FIG. 4. Under normalcellular conditions, NF-κB is sequestered in a complex with IκB. Inresponse to a signal by an unknown pathway, a kinase phosphorylates IκBin the cytoplasm. IκB then degrades which releases the NF-κB. The NF-κBenters the nucleus and is believed to undergo additional phosphorylationby GSK-3β. The phosphorylated NF-κB can then bind to the cellular DNAwhich stimulates transcription of genes encoding cytokines such as IL-1,IL-2, IL-6, IL-8, and TNF-α. It also has a role in stimulatingtranscription of genes which are involved in cell growth such as DNArepair, cell division, and cell survival. The compounds of the aboveformula likely inhibit the phosphorylation of NF-κB by GSK-3 by bindingto the ATP binding pocket of the GSK-3β. As a result, cytokineproduction is inhibited and cell growth is inhibited. The above schemeis to be contrasted with the activity of other compounds such asimidizolines which inhibit NF-κB in the cytoplasm, not in the nucleus.

[0082] The synthesis of the indol-aldisine or indoloazepine skeleton(compounds 2 and 3 shown in FIG. 1) is shown in Scheme 1 and describedin Example 1. It was achieved via a modified route as reported in thesynthesis of aldisine (Annoura and Tatsuoka, Tet. Let. 36: 413-416(1995); Mizuno et al., Chem. Pharm. Bull. 47: 246-256 (1999); Cho etal., J. Heterocycl. Chem. 34: 87-91 (1997); Xu et al., J. Org. Chem. 62:456-464 (1997)).

[0083] Starting with the commercially available 2-indolecarboxylic acid,condensation with the ethyl ester of β-alanine in the presence of EDCIand DMAP, provided the indole 4. Methylation of the indole nitrogen withMeI and K₂CO₃ proceeded in near quantitative yields, rendering theN-methyl indole, which was used for the synthesis of 3. Hydrolysis ofesters 4 and 5 followed by the P₂O₅/MeSO₃H mediated cyclization providedthe key intermediate aldisine derivatives 8 and 9.

[0084] The direct condensation of the aldisine derivatives 8 or 9 withthe imidazolone precursor (Scheme 2) proved to be unsuccessful similarto earlier reports with the pyrrolazepines (Scheme 2) (Prager andTsopelas, Aust. J. Chem. 43: 367-374 (1990); Prager and Tsopelas, Aust.J. Chem. 45: 1771-1777 (1992)). However, TiCl₄ mediated Aldolcondensation with the phenyl oxazolone 12 provided the oxazolonesuccessfully in 55% and 56% yield for compounds 10 and 11, respectively.Treatment of the oxazolone derivatives 10 and 11 with the thiourea underbasic conditions provided the final products, indoloazepines (compounds)2 and 3 in modest yields.

[0085] Hymenialdisine 1 and compounds 2 and 3 were evaluated for theiranti-inflammatory activity by examining the transcriptional activity ofNF-κB in human Jurkat leukemia T-cells and THP-1 cells (human monocyticleukemia cell line) (Examples 2-5). Exposure of human Jurkat leukemiaT-cells to phorbol myristate acetate (PMA/PHA) and THP-1 cells tolipopolysaccharide (LPS), activates NF-κB mediated gene transcription ofseveral pro-inflammatory cytokines, including IL-2, IL-6, IL-8 and TNF-α(Baldwin, Ann. Rev. Immunol. 14: 649-683 (1996); Grilli et al., Int.Rev. Cytol. 143: 1-62 (1993)). The anti-oxidant and non-selective NF-κBinhibitor, pyrrolidinedithiocarbamate (PDTC), has been reported torepress activation of NF-κB and was used as a control in all experimentsin addition to an authentic sample of the natural product,hymenialdisine (Grilli et al., Int. Rev. Cytol. 143: 1-62 (1993); Epinatand Gilmore, Oncogene 18: 6896-6909 (1999); Cuzzocrea et al., Br. J.Pharmacol. 135: 496-510 (2002)). The effect of hymenialdisine andcompounds 2 and 3 on NF-κB's transcriptional activity was evaluated bymeasuring the level of IL-2 and TNF-α production using a competitiveenzyme immunoassay (EIA). The inhibition of IL-2 was evaluated in Jurkatcells after PMA/PHA activation (Mahon and O'Neill, J. Biol. Chem. 270:28557-28564 (1995); Lindgren et al., Molec. Immunol. 38V 267-277(2001)). The inhibition of TNF-α was evaluated in THP-1 cells after LPSactivation (Aikawa et al., Inflamm. Res. 51: 188-194 (2002)). NF-κBmediated transcription of IL-2 production was examined by exposing thecells to PDTC or compounds 1-3, 30 minutes prior to PMA activation.After 24 hours, the cell free supernatant fractions were collected andsubjected to EIA for the quantification of total IL-2 production.

[0086] Compound 2 and the natural product hymenialdisine exhibitedpotent inhibition of IL-2 and TNF-α production, whereas the N-methylcompound 3 had substantially less IL-2 or TNF-α inhibitory activity.Hymenialdisine and compound 2 were found to inhibit DNA binding byNF-κB. Gel electrophoresis assays indicated that none of the compoundshad any notable effect on the DNA binding of the transcription factorAP-1.

[0087] Hymenialdisine and compounds 2 and 3 were tested for inhibitionof the cyclin dependent kinases CDK1 and GSK-3β using the method ofMeijer et al., Chem. Biol. 7: 51-63 (2000). Like hymenialdisine,compound 2 inhibited CDK1 (IC₅₀=0.4 μM) and GSK-3β (IC₅₀=150 ηM) Theinhibitory activity of compound 3 was less pronounced.

[0088] Thus, compounds which have the formula

[0089] wherein R₁, R₂, and R₃ are moieties such that the compoundinhibits NF-κB in human Jurkat Leukemia T-cells activated to transcribeNF-κB mediated gene transcription by phorbol myristate acetate (PMA)provide a new synthetically readily available scaffold for furtheroptimization for cytokine inhibition in animals or humans in needthereof. In a further embodiment, R₁, R₂, and R₃ are moieties such thatthe compound inhibits a cyclin kinase, particularly wherein the cyclinkinase is GSK-3β or CDK-1. In a further embodiment, R₁, R₂, and R₃ areeach independently selected from the group consisting of hydrogen,methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl,hydroxyl, amine, thiol, ester, ether, and amide. It is furtherembodiment, it is preferable that R₃ is hydrogen. In a particularlypreferred embodiment, the compound is compound 2.

[0090] Hymenialdisine and compound 2 were tested for inhibition of thecheckpoint kinases CHK1 and CHK2 by UpState, Inc. Compound 2 inhibitedCHK1 (IC₅₀=237 nM) and CHK2 (IC₅₀=8 nM) and was many folds more potentthan the naturally-occurring hymenialdisines.

[0091] Thus, compounds which have the formula

[0092] wherein R₁, R₂, and R₃ are moieties such that the compoundinhibits checkpoint kinases provide a new synthetically readilyavailable scaffold for further optimization for anticancer therapy inanimals or humans in need thereof. In a further embodiment, R₁₁ R₂, andR₃ are each independently selected from the group consisting ofhydrogen, methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl,acyl, hydroxyl, amine, thiol, ester, ether, and amide. In a furtherembodiment, it is preferable that R₃ is hydrogen. In a particularlypreferred embodiment, the compound is compound 2.

[0093] The present invention further provides a pharmaceuticalcomposition which comprises one or more compounds according to the aboveformulae and a pharmaceutically acceptable carrier, diluent, orexcipient. Thus, the compound may be used in the manufacture of amedicament. Pharmaceutical compositions of the compound synthesizes asdescribed herein can be formulated as solutions or lyophilized powdersfor parenteral administration. The powders can be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation can be a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, standard 5% dextrose in water, or bufferedsodium or ammonium acetate solution. Such formulations are especiallysuitable for parenteral administration, but may also be used for oraladministration or contained in a metered dose inhaler or nebulizer forinsulation. In particular embodiments, it can be desirable to furtheradd one or more excipients selected from the group consisting ofpolyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride, and sodium citrate.

[0094] Alternately, one or more of the above compounds may beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers canbe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Solid carriers include starch, lactose,calcium sulfate dihydrate, terra alba, magnesium stearate or stearicacid, talc, pectin, acacia, agar, or gelatin. Liquid carriers includesyrup, peanut oil, olive oil, saline, and water. The carrier can alsoinclude a sustained release material such as glyceryl monostearate orglyceryl distearate, alone or with a wax. The pharmaceuticalpreparations are made following the conventional techniques of pharmacysuch as milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing, and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension. Such a liquid formulation can be administered directly orfilled into a soft gelatin capsule.

[0095] For rectal administration, one or more of the above compounds canalso be combined with excipients such as cocoa butter, glycerin,gelatin, or polyethylene glycols and molded into a suppository.

[0096] The methods of the present invention further include topicaladministration of one or more of the above compounds. By topicaladministration is meant non-systemic administration, including theapplication of a compound of the invention externally to the epidermis,to the buccal cavity, and instillation into the ear, eye, and nose,wherein the compound does not significantly enter the blood stream. Bysystemic administration is meant oral, intravenous, intraperitoneal, andintramuscular administration. The amount of a compound of the inventionrequired for therapeutic or prophylactic effect upon topicaladministration will, of course, vary with the compound chosen, thenature and severity of the condition being treated and the animal orhuman undergoing treatment, and is ultimately at the discretion of thephysician.

[0097] The topical formulations of the present invention, both forveterinary and for human medical use, comprise one or more of the abovecompounds together with one or more acceptable carriers therefor, andoptionally any other therapeutic ingredients.

[0098] Formulations suitable for topical administration include liquidor semi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required such as: liniments, lotions,creams, ointments, or pastes, and drops suitable for administration tothe eye, ear, or nose.

[0099] Drops according to the present invention may comprise sterileaqueous or oily solutions or suspensions and may be prepared bydissolving one or more of the above compounds in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution can then be clarified by filtration, transferredto a suitable container which is then sealed and sterilized byautoclaving or maintaining at 90-100° C. for half an hour.Alternatively, the solution can be sterilized by filtration andtransferred to the container by an aseptic technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the dropsare phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

[0100] Lotions according to the present invention include those suitablefor application to the skin or eye. An eye lotion can comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

[0101] Creams, ointments, or pastes according to the present inventionare semi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the compound in finely-dividedor powdered form, alone or in solution or suspension in an aqueous ornon-aqueous fluid, with the aid of suitable machinery, with a greasy ornon-greasy basis. The basis may comprise hydrocarbons such as hard, softor liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; anoil of natural origin such as almond, corn, arachis, castor or oliveoil; wool fat or its derivatives, or a fatty acid such as stearic oroleic acid together with an alcohol such as propylene glycol ormacrogols. The formulation may incorporate any suitable surface activeagent such as an anionic, cationic or non-ionic surface active agentsuch as sorbitan esters or polyoxyethylene derivatives thereof.Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredientssuch as lanolin, may also be included.

[0102] The present invention also provides methods of treatment ofdiseases associated with NF-κB activation, which methods compriseadministering to an animal, particularly an animal, most particularly ahuman in need thereof, one or more of the above compounds. The presentinvention particularly provides methods for treating inflammatorydisorders; particularly rheumatoid arthritis, inflammatory boweldisease, and asthma; dermatosis, including psoriasis and atopicdennatitis; autoimmune diseases; tissue and organ rejection; Alzheimer'sdisease; stroke; atherosclerosis; restenosis; cancer, including Hodgkinsdisease; and certain viral infections, including AIDS; osteoarthritis;osteoporosis; and Ataxia Telangiectasia.

[0103] For acute therapy, parenteral administration of one or more ofthe above compounds is preferred. An intravenous infusion of thecompound can be in 5% dextrose in water or normal saline, or a similarformulation with suitable excipients. Typically, the parenteral dosewill be at a concentration effective to inhibit activation of NF-κB. Theprecise amount of an inventive compound which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect.

[0104] The compounds of the above formula can also be administeredorally to the animal or human, in a manner such that the concentrationof drug is sufficient to inactivate NF-κB or to achieve any othertherapeutic indication as disclosed herein. Typically, a pharmaceuticalcomposition containing one or more of the above compounds isadministered in amount which for the body weight of the animal or humaninactivates the NF-κB.

[0105] One or more of the above compounds can also be administeredtopically to the animal or human, in a manner such that theconcentration of drug is sufficient to inhibit NF-κB.

[0106] In some embodiments of the invention, the compound of the aboveformula is used in combination with one or more other anti-inflammatory,anti-viral, anti-fungal, amoebicidal, trichomonocidal, analgesic,anti-neoplastic, anti-hypertensives, anti-microbial and/or steroid drugsor potentiators. Such drugs include triprolidine or its cis-isomer whichis used in combination with chemotherapeutic agents; a compound of theabove formula and procodazole, 1H-benzimidazole-2-propanoic acid;[β-(2-benzimidazole) propionic acid 2-(2-carboxyethyl)benzimidazole;propazol] which is a non-specific immunoprotective agent active againstviral and bacterial infections that is used with the compound of theabove formula; or a compound of the above formula and aplatinum-containing drug such as cisplatin which binds DNA whichinterferes with its DNA repair mechanism and thereby causing cellulardeath. Other drugs which can be used with a compound of the aboveformula, and optionally another chemotherapeutic agent, in the methodsof the invention include macrophage colony-stimulating factor (M-CSF),7-thia-8-oxoguanosine, 6-mercaptopurine, vitamin A (retinol), and otherknown anti-tumor potentiators which can be used in conjunction with thecompounds of the above formula include, monensin, an anti-senseinhibitor of the RAD51 gene, bromodeoxyuridine, dipyridamole,indomethacin, a monoclonal antibody, an anti-transferrin receptorimmunotoxin, metoclopramide,N-solanesyl-N,N′-bis(3,4-dimethoxybenzyl)ethylenediamine, leucovorin,heparin, N-[4-[(4-fluorphenyl)sulfonly]phenyl]acetamide, heparinsulfate, cimetidine, a radiosensitizer, a chemosensitizer, a hypoxiccell cytotoxic agent, muramyl dipeptide, vitamin A, 2′-deoxycoformycin,a bis-diketopiperazine derivative, and dimethyl sulfoxide otheranti-tumor potentiators.

[0107] The chemotherapeutic agents which can be used with a compound ofthe above formula and an optional potentiator are generally grouped asDNA-interactive agents, antimetabolites, tubulin-interactive agents,hormonal agents, and others such as asparaginase or hydroxyarea. Each ofthe groups of chemotherapeutic agents can be further divided by type ofactivity or compound.

[0108] DNA-interactive agents include the alkylating agents, forexample, cisplatin, cyclophosphamide, altretamine; the DNAstrand-breaking agents, such as bleomycin; the intercalatingtopoisomerase II inhibitors, for example, dactinomycin and doxorubicin;the nonintercalating topoisomerase II inhibitors, such as etoposide andteniposide; and the DNA minor groove binder plicamycin.

[0109] The alkylating agents form covalent chemical adducts withcellular DNA, RNA, and protein molecules and with smaller amino acids,glutathione and similar chemicals. Generally, these alkylating agentsreact with a nucleophilic atom in a cellular constituent, such as anamino, carboxyl, phosphate, sulfhydryl group in nucleic acids, proteins,amino acids, or glutathione. The mechanism and the role of thesealkylating agents in cancer therapy is not well understood. Typicalalkylating agents include: nitrogen mustards, such as chlorambucil,cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard;aziridine such as thiotepa; methanesulphonate esters such as busulfan;nitroso ureas, such as carmustine, lomustine, streptozocin; platinumcomplexes, such as cisplatin, carboplatin; bioreductive alkylator, suchas mitomycin, and procarbazine, dacarbazine, and altretamine.

[0110] DNA strand breaking agents include Bleomycin. DNA topoisomeraseII inhibitors include the following: intercalators, such as amsacrine,dactinomycin, daunorubicin, doxorubicin, idarubicin, and mitoxantrone;and nonintercalators, such as etoposide and teniposide. The DNA minorgroove binder is Plicamycin.

[0111] The antimetabolites interfere with the production of nucleicacids by one or the other of two major mechanisms. Some of the drugsinhibit production of the deoxyribonucleoside triphosphates that are theimmediate precursors for DNA synthesis, thus inhibiting DNA replication.Some of the compounds are sufficiently like purines or pyrimidines to beable to substitute for them in the anabolic nucleotide pathways. Theseanalogs can then be substituted into the DNA and RNA instead of theirnormal counterparts. The antimetabolites useful herein include: folateantagonists such as methotrexate and trimetrexate; pyrimidineantagonists, such as fluorouracil, fluorodeoxyunridine, CB3717,azacitidine and floxuridine; purine antagonists such as mercaptopurine,6-thioguanine, pentostatin; sugar modified analogs such as cytarabineand fludarabine; and ribonucleotide reductase inhibitors such ashydroxyurea.

[0112] Tubulin interactive agents act by binding to specific sites ontubulin, a protein that polymerizes to form cellular microtubules.Microtubules are critical cell structure units. When the interactiveagents bind on the protein, the cell can not form microtubules tubulininteractive agents include colchicine, vincristine and vinblastine, bothalkaloids and paclitaxel and cytoxan.

[0113] Hormonal agents are also useful in the treatment of cancers andtumors. They are used in hormonally susceptible tumors and are usuallyderived from natural sources. These include estrogens, conjugatedestrogens and ethinyl estradiol and diethylstilbesterol; chlortrianisenand idenestrol; progestins such as hydroxyprogesterone caproatemedroxyprogesterone, and megestrol; and androgens such as testosterone,testosterone propionate, fluoxymesterone, and methyltestosterone.

[0114] Adrenal corticosteroids are derived from natural adrenal cortisolor hydrocortisone. They are used because of their anti inflammatorybenefits as well as the ability of some to inhibit mitotic divisions andto halt DNA synthesis. These compounds include, prednisone,dexamethasone, methylprednisolone, and prednisolone.

[0115] Leutinizing hormone releasing hormone agents orgonadotropin-releasing hormone antagonists are used primarily thetreatment of prostate cancer. These include leuprolide acetate andgoserelin acetate. They prevent the biosynthesis of steroids in thetestes.

[0116] Antihormonal antigens include: antiestrogenic agents such astamoxifen; antiandrogen agents such as flutamide; and antiadrenal agentssuch as mitotane and aminoglutethimide.

[0117] Hydroxyurea, which appears to act primarily through inhibition ofthe enzyme ribonucleotide reductase, can also be used in combinationwith the compound of the above formula.

[0118] Asparaginase is an enzyme which converts asparagine tononfunctional aspartic acid and thus blocks protein synthesis in thetumor. Asparaginase can also be used in combination with the compound ofthe above formula to treat cancer.

[0119] Other chemotherapeutic benzimidazoles and griseofulvin can alsobe used in combination with the compound of the above formula andoptionally a potentiator to treat or inhibit the growth of cancer orextend the life span of a animal or human having cancer.

[0120] The amount and identity of a chemotherapeutic agent that is usedwith a compound of the above formula in the methods of the inventionwill vary according to cellular response, patient response andphysiology, type and severity of side effects, the disease beingtreated, the preferred dosing regimen, patient prognosis, or other suchfactors.

[0121] The compound of the above formula can be used in combination withone or more other agents or combination of agents known to possessanti-leukemia activity including, by way of example, α-interferon;interleukin-2; cytarabine and mitoxantrone; cytarabine and daunorubicinand 6-thioguanine; cyclophosphamide and 2-chloro-2′-deoxyadenosine;VP-16 and cytarabine and idorubicin or mitoxantrone; fludarabine andcytarabine and γ-CSF; chlorambucil; cyclophosphamide and vincristine and(prednisolone or prednisone) and optionally doxorubicin; tyrosine kinaseinhibitor; an antibody; glutamine; clofibric acid; all-trans retinoicacid; ginseng diyne analog; KRN8602 (anthracycline drug); temozolomideand poly(ADP-ribose) polymerase inhibitors; lysofylline; cytosinearabinoside; chlythorax and elemental enteral diet enriched withmedium-chain triglycerides; amifostine; gilvusmycin; or a hot waterextract of the bark of Acer nikoense.

[0122] The compounds of the above formula can further be administered toan animal or human with one or more imidazolines and optionally one ormore of the above drugs or potentiators as a treatment for cellularproliferative diseases. As used herein, antiproliferative agents arecompounds, which induce cytostasis or cytotoxicity. Cytostasis is theinhibition of cells from growing while cytotoxicity is defined as thekilling of cells. Specific examples of antiproliferative agents includeantimetabolites, such as methotrexate, 5-fluorouracil, gemcitabine,cytarabine; anti-tubulin protein agents such as the vinca alkaloids,paclitaxel, colchicine; hormone antagonists, such as tamoxifen, LHRHanalogs; and nucleic acid damaging agents such as the alkylating agentsmelphalan, BCNU, CCNU, thiotepa, intercalating agents such asdoxorubicin and metal coordination complexes such as cisplatin andcarboplatin.

[0123] The following examples are intended to promote a furtherunderstanding of the present invention.

EXAMPLE 1

[0124] This example shows the synthesis of compounds 2 and 3 as shown inFIGS. 1B and 1C and Scheme 1.

[0125] Reactions were carried out in oven-dried glassware under nitrogenatmosphere, unless otherwise noted. All commercial reagents were usedwithout further purification. All solvents were reagent grade. THF wasfreshly distilled from sodium/benzophenone under nitrogen. CH₂Cl₂ wasfreshly distilled from CaH₂ under nitrogen. All reactions weremagnetically stirred and monitored by thin layer chromatography withAnaltech 0.25-mm pre-coated silica gel plates (Analtech, Inc., Newark,Del.). Column chromatography was carried out on silica gel 60 (230-400mesh) supplied by EM Science (END Chemicals, Inc., Gibbstown, N.J.).

[0126] Yields refer to chromatographically and spectroscopically purecompounds unless otherwise stated. Infrared spectra were recorded on aNicolet IR/42 spectrometer. Proton and carbon NMR spectra were recordedon a Varian Gemini-300 spectrometer or a Varian VXR-500 spectrometer(Varian Medical Systems, Inc., Palo Alto, Calif.). Chemical shifts werereported relative to the residue peaks of solvent chloroform (δ 7.24 for¹H and δ 77.0 for ¹³C) and dimethyl sulfoxide (δ 2.49 for ¹H and δ 39.5for ¹³C). High-resolution mass spectra were obtained at the MassSpectrometry Laboratory of the University of South Carolina, Departmentof Chemistry & Biochemistry with a Micromass VG-70S mass spectrometer.Gas chromatography/low-resolution mass spectra were recorded on aHewlett Packard 5890 Series II gas chromatograph connected to a TRIO-1EI mass spectrometer. All regents were obtained from Aldrich ChemicalCo., St. Louis, Mo., and used as received.

[0127] Synthesis of 3-[(1H-indole-2-carbonyl)-amino]-propionic acidethyl ester, compound 4, was as follows. To a mixture of2-indolecarboxylic acid (161 mg, 1.0 mmol), DMAP (200 mg, 6 mmol) andalanine ethyl ester hydrochloride (168 mg, 1.1 mmol) in 10 mL anhydrousCH₂Cl₂ was added EDCI (215 mg, 1.1 mmol) at 0° C. The mixture then wasstirred at 0° C. for 4 hours and at room temperature for 20 hours. Themixture was washed with water (20 mL) and 10% HCl (10 mL), dried overanhydrous Na₂SO₄, filtered, and concentrated to yield product 4 as awhite solid (230 mg, 88.5%). Mp 158-160° C. ¹H NMR:

[0128] MHz, CDCl₃) δ 1.28 (t, 3H, J=7.2 Hz), 2.69 (t, 2H, J=5.7 Hz),3.78 (q, 2H, J=6.0 Hz), 4.18 (q, 2H, J=6.9, 7.2 Hz), 6.89 (s, 1H), 7.05(s, 1H), 7.13-7.68 (m, 4H), 9.86 (s, 1H). ¹³C NMR: (74.47 MHz, CDCl₃) δ14.1, 33.9, 34.9, 60.8, 102.2, 111.9, 120.4, 121.8, 124.3, 127.5, 130.5,136.4, 161.7, 172.8. IR: (NaCl) 3377, 3352, 1716, 1624, 1552, 1325,1207, 748 cm⁻¹. HRMS m/e calcd for C₁₄H₁₆N₂O₃ (M) 260.1161, found260.1159.

[0129] Synthesis of 3-[(1-methyl-1H-indole-2-carbonyl)-amino]-propionicacid ethyl ester, compound 5, was as follows. A solution of compound 4(0.5 g, 1.9 mmol), K₂CO₃ (1.06 g, 7.68 mmol) and iodomethane (0.29 mL,4.64 mmol) in acetonitrile (15 mL) was stirred at reflux for 24 hours.The mixture was cooled, filtered and the filtrate was evaporated invacuo. The crude material was further purified by column chromatographyon silica gel (CH₂Cl₂-AcOEt 3:1) to yield compound 5 as white solid (512mg, 98%); Mp 75-77° C. ¹H NMR: (300 MHz, CDCl₃) δ 1.28 (t, 3H, J=7.2Hz), 2.67 (m, 2H), 3.72 (m, 2H), 4.07 (s, 3H), 4.22 (q, 2H, J=6.9 Hz),6.86 (s, 1H), 7.05-7.65 (m, 4H). ¹³C NMR: (74.47 MHz, CDCl₃) δ 14.1,31.4, 33.9, 34.8, 60.7, 103.8, 110.1, 120.3, 121.7, 123.9, 125.9, 131.8,138.9, 162.4, 172.6. IR (NaCl): 3263, 1732, 1630, 1552, 1462, 1392,1321, 1182, 746 cm⁻¹. HRMS m/e calcd for C₁₅H₁₈N₂O₃ (M) 274.1317, found274.1315.

[0130] Synthesis of 3-[(1-methyl-1H-indole-2-carbonyl)-amino]-propionicacid, compound 7, was as follows. A solution of compound 5 (420 mg, 1.53mmol) and LiOH (129 mg, 3.07 mmol) in ethanol (14 mL) was stirred atroom temperature for 18 hours. After cooling, the solvent was evaporatedin vacuo. The residue was dissolved in water and acidified with 1N HCLto pH=3 to afford white precipitate, which isolated by filtration andwashed with water to provide compound 7 (360 mg, 95%); Mp 160-162° C. ¹HNMR: (300 MHz, d⁶-DMSO) δ 2.49 (t, 2H, J=7.2 Hz), 3.46 (q, 2H, J=6.6Hz), 3.97 (s, 3H), 7.07-7.64 (m, 5H), 8.54 (t, 1H, J=5.7 Hz). ¹³C NMR:(74.47 MHz, d⁶-DMSO) δ 31.3, 33.8, 35.2, 104.3, 110.5, 120.1, 121.5,123.5, 125.6, 132.1, 138.4, 161.9, 173.0. IR (NaCl): 3385, 2966, 1713,1603, 1550, 1468, 1425, 1400, 1282, 1228, 1192, 910, 748 cm⁻¹. HRMS m/ecalcd for C₁₃H₁₄N₂O₃ (M) 246.1004, found 246.1016.

[0131] Synthesis of10-methyl-3,4-dihydro-2H,10H-azepino[3,4-b]indole-1,5-dione, compound 9,was as follows. Compound 7 (2.16 g, 8.78 mmol) was added to a clearsolution of P₂O₅ (3.49 g, 12.3 mmol) in MeSO₃H (21 mL) at 60° C. Themixture was heated to 110° C. for 1 hour, after which the mixture wascooled to room temperature. The reaction mixture was poured intoice-water, stirred for 30 minutes, filtered and washed with water, toprovide compound 9 (1.143 g) as a solid (57%). Mp 200-205° C. ¹H NMR:(500 MHz, d⁶-DMSO) δ 2.77 (t, 2H, J=5.5 Hz), 3.39 (t, 2H, J=5.5, 4.5Hz), 3.99 (s, 3H), 7.27 (t, 1H, J=7.0, 8.0 Hz), 7.37 (t, 1H, J=7.5 Hz),7.62 (d, 1H, J=8.0 Hz), 8.27 (d, 1H, J=8.0 Hz), 8.77 (t, 1H, J=5.0, 6.0Hz); ¹³C NMR: (124.1 MHz, d⁶-DMSO) δ 33.0, 37.3, 45.6, 111.6, 115.5,123.4, 123.8, 125.3, 125.7, 135.4, 138.7, 163.1, 196.4. IR (NaCl): 3204,3000, 2924, 1662, 1641, 1506, 1473, 1371, 724 cm⁻¹; HRMS m/e calcd forC₁₃H₁₂N₂O₂ (M) 228.0899, found 228.0887.

[0132] Synthesis of10-methyl-5-(5-oxo-2-phenyl-oxazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 11 was as follows. A solution of TiCl₄ (1.54 mL, 14 mmol) inCH₂Cl₂ (10 mL) was added into THF (10 mL) at 0° C. Compound 9 (802 mg,3.5 mmol) and 2-phenylazlactone (1.13 g, 7 mmol) were added to thismixture. The mixture was stirred at 0° C. for 20 minutes after whichpyridine (1.13 mL, 14 mmol) was added. The reaction mixture was stirredat 0° C. for an additional 2 hours, after which it was allowed to stirovernight at room temperature. NH₄Cl (80 mL, sat solution in water) wasadded and the mixture was stirred for 10 minutes. The mixture wassubsequently extracted with ethyl acetate (3 times), the extractscombined, dried with anhydrous Na₂SO₄₁ filtered, concentrated and theresidue was purified with column chromatography on silica gel (ethylacetate-hexane 8:2) to yield compound 11 (0.73 g, 56%) as yellow solid.Mp: 189-192° C. ¹H NMR: (300 MHz, CDCl₃) δ 3.54 (t, 2H, J=3.3, 3.6 Hz),3.59 (t, 2H, J=2.7, 3.3 Hz), 4.09 (s, 3H), 6.94 (s, 1H), 7.24-7.27 (m,1H), 7.38-7.49 (m, 5H), 7.78 (d, 1H, J=4.8 Hz), 7.92 (d, 2H, J=6.0 Hz);¹³C NMR: (74.47 MHz, CDCl₃) δ 31.9, 38.2, 38.4, 110.1, 115.8, 121.3,124.3, 125.0, 125.1, 125.8, 127.5, 128.7, 130.0, 131.7, 132.4, 138.5,144.5, 159.4, 165.3, 165.9. IR: (NaCl) 1784, 1753, 1662, 1633, 1473cm⁻¹; LRMS (EI): 371.1(M); HRMS m/e calcd for C₂₂H₁₇N₃O₃ (M) 371.1270,found 371.1268.

[0133] Synthesis of5-(2-amino-5-oxo-1,5-dihydro-imidazol-4-ylidene)-10-methyl-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 3, was as follows. LiH (48 mg, 6 mmol) was dissolved in ethanol(150 mL) and to this solution was added compound 11 (371 mg, 1.0 mmol)and S-benzylisothiouronium chloride (1.01 g, 5 mmol). The reaction wasrefluxed 48 hours, after which the solvent was evaporated in vacuo. 50mL ethanol was added to the reaction and was evaporated. This wasrepeated 3 times. Ethanol (15 mL) was added again and the reaction wasrefluxed for 3 hours, after which the solvent was distilled off invacuum. 1N HCL(aq) (50 mL) was added and the product was extracted withn-butanol (3×50 mL). The extracts were combined and washed with brine(3×20 mL), dried, concentrated and the product was purified by columnchromatography on silica gel (CH₂Cl₂: MeOH:NH₃.H₂O, 3:1:0.1) to yieldcompound 3 as light yellow solid (96 mg, 31%) Mp: >260° C. ¹H NMR: (300MHz, d⁶-DMSO) δ 2.99-3.48 (m, 4H) 3.92 (s, 3H), 7.19 (t, 1H, J=7.2 Hz),7.36 (t, 1H, J=6.6 Hz), 7.55 (d, 1H, J=8.1 Hz), 7.65 (d, 1H, J=8.4 Hz),8.57 (t, 1H, J=5.1 Hz), 9.15-9.25 (br-s,1H), 10.48 (s, 1H); ¹³C NMR:

[0134] MHz, d⁶-DMSO) δ 32.1, 37.1, 38.4, 111.7, 112.6, 122.1, 122.2,123.5, 123.7, 125.1, 128.1, 132.8, 138.5, 154.6, 163.0, 165.1; IR:(KBr-pellet) 3211, 1699, 1635 1508, 1477 cm⁻¹; LRMS (EI): 308.7 (M);HRMS m/e calcd for C₁₆H₁₅N₅O₂ (M) 309.1304, found 309.1291.

[0135] Synthesis of 3-[(1H-indole-2-carbonyl)-amino]-propionic acidethyl ester, compound 6, was as follows.

[0136] A mixture of compound 4 (1.838 g, 7.38 mmol) and LiOH (0.6 g,14.1 mmol) was stirred at room temperature in ethanol (50 mL) for 20hours, the ethanol was evaporated to dryness in vacuo. The residue wasdissolved in water (50 mL), acidified the solution to pH=1 with HCL(aq), at which a white solid precipitated. The mixture was allowed tostand at 0° C. for 30 minutes, after which the product was filtered of,dried in vacuum, to yield compound 6 (1.45 g, 84%). Mp 232° C. ¹H NMR:(300 MHz, d⁶-DMSO) δ 2.52 (t. 2H, J=6.9 Hz), 3.47 (q, 2H, J=6.6, 6.0Hz), 6.98 (t, 1H, J=7.2 Hz), 7.10 (s, 1H), 7.13 (t, 1H, J=7.2 Hz), 7.41(1H, J=8.1 Hz), 7.57 (1H, J=8.1 Hz), 8.52 (s, 1H), 11.55 (s, 1H), 12.25(s, 1H); ¹³C NMR: (74.47 MHz) (d⁶-DMSO) δ 34.6, 35.9, 103.2, 112.9,120.3, 122.1, 123.9, 127.8, 132.4, 137.1, 161.8, 173.4; IR (NaCl): 3422,3273, 1745, 1707, 1643, 1549, 1417, 1341, 1259, 746 cm⁻¹; HRMS m/e calcdfor C₁₂H₁₂N₂O₃ (M) 232.0848, found 232.0844.

[0137] Synthesis of 3,4-dihydro-2H,10H-azepino[3,4-b]indole-1,5-dione,compound 8, was as follows. Compound 6 (116 mg, 0.5 mmol) was added to aclear solution of P₂O₅ (232 mg, 0.8 mmol) in MeSO₃H (1.57 mL) at 60° C.The mixture was heated to 110° C. for 1.5 hour, after which the mixturewas cooled to room temperature. The reaction mixture was poured intoice-water, stirred for 30 minutes, filtered, dissolved in acetone (100mL), filtered again, and the filtrate was concentrated. The product wasfurther purified by column chromatography on silica gel to yieldcompound 8 (88 mg, 82%) Mp: 257-260° C. ¹H NMR (300 MHz, d⁶-DMSO) δ2.80-2.85 (m, 2H), 3.40-3.46 (m, 2H), 7.22-7.38 (m, 2H), 7.51 (d, 1H,J=9.0 Hz), 8.28 (d, 1H, J=9 Hz), 8.72 (m, 1H), 12.41 (s, 1H); ¹³C NMR(74.47 MHz, d⁶-DMSO) δ 36.7, 44.2, 112.7, 113.8, 122.8, 122.9, 125.0,126.2, 134.7, 135.7, 162.5, 195.3; IR: (KBr-pellet) 3209, 1664, 1630,1523, 1437, 1408 cm⁻¹; LRMS (EI): M⁺=214.3; HRMS m/e calcd forC₁₂H₁₀N₂O₂ (M) 214.0742, found 214.0740.

[0138] Synthesis of5-(5-oxo-2-phenyl-oxazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 10, was as follows. A solution of TiCl₄ (132 μL, 1.2 mmol) inCH₂Cl₂ (1.2 mL) and the mixture was added into THF (3 mL) at 0° C.Compound 8 (65 mg, 0.3 mmol) and 2-phenylazlactone (97 mg, 0.6 mmol)were subsequently added to this mixture. The reaction mixture wasstirred at 0° C. for an additional 2 hours, after which it was allowedto stir overnight at room temperature. NH₄Cl (8 mL, sat. solution inwater) was added and the mixture was stirred for 10 minutes. The mixturewas subsequently extracted with ethyl acetate (3 times), the extractscombined, dried with anhydrous Na₂SO₄₁ filtered, concentrated and theresidue was purified with column chromatography on silica gel (acetone)to yield compound 10 (59 mg, 55%) as a foamy yellow solid. Mp: 247-250°C. ¹H NMR (300 MHz, d⁶-DMSO) δ 3.36-3.47 (m, 4H), 7.14 (t, 1H, J=8.1Hz), 7.30 (t, 1H, J=9.0 Hz), 7.49-7.58 (m, 4H), 7.81-7.87 (m, 3H),8.45-8.56 (m, 1H), 12.11(s, 1H); ¹³C NMR (74.47 MHz, d⁶-DMSO) δ 37.7,38.6, 112.9, 115.3, 121.0, 125.1, 125.3, 126.5, 127.4, 129.0, 129.8,133.2, 134.0, 136.9, 146.3, 158.4, 165.0, 166.1; IR: (KBr-pellet) 3358,3179, 1749, 1653, 1633, 1448 cm⁻¹; HRMS m/e calcd for C₂₁H₁₅N₃O₃ (M)357.1113, found 357.1106.

[0139] Synthesis of5-(2-amino-5-oxo-1,5-dihydro-imidazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 2, was as follows. LiH (16 mg, 2 mmol) was dissolved in ethanol(60 mL) and to this solution was added compound 10 (150 mg, 0.4 mmol)and S-benzylisothiouronium chloride (405 mg, 2 mmol). The reaction wasrefluxed 48 hours after which the reaction was cooled and the solventwas evaporated in vacuo. Ethanol (20 mL) added and subsequentlyevaporated 3 times. Ethanol (5 mL) was added again and the reaction wasrefluxed for 3 hours. The solvent was evaporated off in vacuum, 1NHCl_((aq)) (15 mL) was added and the mixture was extracted withn-butanol (3×15 mL), the extracts were washed with brine (3×10 mL),dried, concentrated and the product was purified by columnchromatography on silica gel (CH₂Cl₂: MeOH:NH₃.H₂O, 3:1:0.1) to yieldcompound 2 (35 mg, 30%) as light yellow solid, Mp:>260° C.; ¹H NMR (300MHz, d⁶-DMSO) δ 3.20-3.40 (br., 4H), 7.16 (t, 1H, J=12.0 Hz), 7.29 (t,1H, J=12.0 Hz), 7.52 (m, 2H), 8.30-8.50 (m, 2H), 9.05-9.25 (br., 1H),10.30-10.40 (m, 1H), 12.48 (s, 1H); ¹³C NMR: (124.1 MHz), d⁶-DMSO) δ36.5, 39.2, 112.7, 113.5, 121.9, 122.3, 122.8, 124.5, 125.0, 128.6,132.8, 137.0, 154.6, 163.4, 165.5; IR: (KBr-pellet) 3294, 1620, 1475,1251 cm¹; LRMS (EI): 295.1 (M); HRMS (FAB) calcd for C₁₅H₁₄N₅O₂ (M+H)296.1148, found 296.1144.

EXAMPLE 2

[0140] This example shows that compound 2, like hymeniadisine 1,exhibits a significant dose response inhibition of IL-2 production whenmeasured in a competitive enzyme immunoassay (EIA) for IL-2 expression.

[0141] Human Jurkat leukemia T-cells (clone E6-1; ATCC No. TIB-152,American Type Culture Collection, 10801 University Boulevard, Manassas,Va.) are grown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.)supplemented with 10% fetal bovine serum, penicillin (614 ηg/mL),streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. Toeach well of a flat bottomed 96 well culture plate 0.2 mL 1×10⁶ JurkatE6-1 cells/mL were added. Each sample was then treated in duplicate withthe compounds in DMSO at either 10 μM, 1 μM, 0.1 μM or 10 ηM and allowedto incubate for thirty minutes at 37° C., 5% CO₂. Cell free supernatantfractions were collected from stimulated cultures incubated for 24 hr at37° C., 5% CO₂. Cultures were stimulated with phytohemagglutinin (PHA,Sigma-Aldrich, St. Louis, Mo.) at 1 μg/mL; and phorbol myristate acetate(PMA, Sigma-Aldrich, St. Louis, Mo.) at 50 ng/mL. The concentration ofIL-2 in each sample was then measured using a competitive enzymeimmunoassay (Neogen Corporation, Lansing, Mich.) according to themanufacturer's protocol. Known IL-2 concentrations were plotted and fita 4 parameter logistic curve. Unknown concentrations were thenextrapolated from the standard curve.

[0142] Hymenialdisine 1 exhibited significant inhibition of IL-2production with an IC₅₀ value of 2.4 μM (Table 1 and FIG. 2B). Treatmentof the Jurkat cells with compound 2 at concentrations ranging from 0.1μM to 10 μM exhibited also a significant dose response inhibition ofIL-2 production (FIG. 2C). Compound 2 exhibited an inhibition of IL-2production with an IC₅₀ value of 3.5 μM.

[0143] Interestingly, blockage of the N-indole position with a methylgroup, compound 3, indicated no significant inhibition of IL-2production up to the concentration tested (FIG. 2D, measured up to 10μM). This further supports the significance of the pyrrolic-H-moiety inpotential hydrogen bonding interactions (Meijer et al., Chem. Biol. 7:51-63 (2000)). FIG. 2A shows the effect of PDTC on the cells. TABLE 1Inhibition of IL-2 production measured by EIA for PDTC, hymenialdisine 1and compounds 2 and 3 in PMA/PHA-activated Jurkat T cells. Inhibition ofIL-2 Compounds Production IC₅₀, (μM)^(a) PDTC (control) 5.123 (±0.407)Hymenialdisine 1 2.411 (±0.710) Compound 2 3.547 (±0.092) Compound 3  >10

EXAMPLE 3

[0144] This example shows that compound 2, like hymeniadisine 1,exhibits a significant inhibition of NF-κB-DNA binding when measured inan EMSA assay for NF-κB-DNA binding in which the nuclear extracts ofPMA/PHA activated Jurkat cells were examined for inhibition of DNAbinding of NF-κB by compounds 1-3 using gel electrophoresis (EMSA).

[0145] Human Jurkat leukemia T-cells (clone E6-1; ATCC No. TIB-152,American Type Culture Collection, 10801 University Boulevard, Manassas,Va.) are grown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.)supplemented with 10% fetal bovine serum, penicillin (614 ηg/mL),streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. TheJurkat cells 1×10⁶ cells/mL) are subsequently treated with variousconcentrations of the compounds in DMSO for 30 min. at 37° C. and 5% CO₂followed by PMA (50 ng/mL) and PHA (1 μM/mL) stimulation for anadditional 30 minutes. The cells were harvested by centrifugation,washed in ice cold PBS and the nuclear extracts were prepared aspreviously described (Dignam et al., Nucl. Acids Res. 11: 1475-1489(1983)). The protein concentration of the extracts was determinedaccording to the Method of Bradford (1976) with BiORad reagents.

[0146] Nuclear extracts were incubated for 20 minutes at roomtemperature with a double stranded Cy3 labeled NF-κB consensusoligonucleotide, 5′-AGTTGAGGGGACTTTC CCAGGC-3′ (SEQ ID NO:1). Thebinding mixture (25 μL) contained 10 mM HEPES-NaOH pH 7.9, 4 mMTris-HCL, pH 7.9, 6.0 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.3mg/mL bovine serum albumin, and 1 μg of poly (dI:dC). The bindingmixtures (10 μg of nuclear extract protein) were incubated for 20minutes at room temperature with 0.16 pmol of Cy3 labeledoligonucleotide. The mixture was loaded on a 4% polyacrylamide gelprepared in 1×TRIS borate/EDTA buffer and was electrophoresed at 200 Vfor 20 minutes. After electrophoresis the gel was analyzed using aphosphorimager (BIORAD FX PLUS, Biorad Laboratories, Inc., Hercules,Calif.) for detection of NF-κB-DNA binding.

[0147] The results are shown in FIG. 3 and Table 2. Control lanesincluded lane 1, in which the NF-κB p65 antibody was used to confirm theNF-κB-DNA complex (FIG. 3). Addition of the antibody to the nuclearextract of activated Jurkat cells resulted in a supershift indicated inFIG. 3 (lane 1). The DNA-NF-κB complex is illustrated in lane 2. Lane 3includes the DNA binding of a non stimulated cell extract, which asanticipated resulted in no significant band shift (lane 3). Lanes 4 and5, were treated with 5 μM and 1 μM PDTC, respectively as a positivecontrol for NF-κB-DNA binding inhibition.

[0148] Inhibition of NF-κB-DNA binding was found to be 51% and 30% for 5μM and 1 μM PDTC, respectively (lanes 4 and 5) as compared to theactivated control (lane 2). Hymenialdisine at 5 μM (46% inhibition) and1 μM concentration (3% inhibition, lanes 6 and 7, respectively)indicated significant inhibition of DNA binding as reported earlier(Roshak et al., J. Pharmacol. Exp. Ther. 283: 955-961 (1997)). Compound2, demonstrated similar inhibition of NF-κB-DNA binding as the naturalproduct hymenialdisine with 49% and 22% inhibition at 5 μM and 1 μM,respectively (lanes 8 and 9). Compound 3 did not appear to substantiallyinhibit DNA binding as measured up to concentrations of 5 μM (lanes 10and 11). In fact, a small increase in binding was observed as comparedto the activated control. A summary of the percent inhibition relativeto the activated control is listed in Table 3. Similar to the studiesreported on hymenialdisine (Roshak et al., J. Pharmacol. Exp. Ther. 283:955-961 (1997)), no significant inhibition of DNA binding was observedwhen compounds 1-3 were tested for inhibition of DNA binding with thetranscription factor AP-1. TABLE 2 Inhibition of NF-κB-DNA bindingmeasured by EMSA for PDTC, hymenialdisine 1 and compounds 2 and 3 inPMA- activated Jurkat cells. Inhibition of DNA-binding (relative toCount per activated mm² NF-κB Compounds control) bands Activated cells —105.55 (control) 5.0 μM PDTC (control) 51% 51.68 1.0 μM PDTC (control)30% 73.48 5.0 μM Hymenialdisine 1 46% 57.06 1.0 μM Hymenialdisine 1  3%102.14 5.0 μM Compound 2 49% 53.40 1.0 μM Compound 2 22% 82.38 5.0 μMCompound 3  0% 121.76 1.0 μM Compound 3  0% 118.82

EXAMPLE 4

[0149] This example shows that compound 2, like hymenialdisine 1,exhibits a significant dose response inhibition of TNF-α when measuredin a competitive enzyme immunoassay (EIA) for TNF-α expression. Theassay measured the ability of the compounds to inhibit TNF-α productionin LPS stimulated THP-1 cells (Aikawa et al., Inflamm. Res. 51V 188-194(2002)).

[0150] THP-1 cells are grown in RPMI-1640 Media (Gibco-BRL, Rockville,Md.) supplemented with 5% fetal bovine serum, penicillin (614 ηg/mL),streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. Toeach well of a flat bottomed 96 well culture plate 0.2 mL 1×10⁶ THP-1cells/mL were added. Each sample was then treated in duplicate with thecompounds in DMSO at either 10 μM, 1 μM, 0.1 μM or 10 ηM and allowed toincubate for thirty minutes at 37° C., 5% CO₂. Cell free supernatantfractions were collected from stimulated cultures incubated for 3 hr at37° C., 5% CO₂. Cultures were stimulated with LPS (Sigma-Aldrich, St.Louis, Mo.) at 1 μg/mL. The concentration of TNF-α in each sample wasthen measured using a competitive enzyme immunoassay (NeogenCorporation, Lansing, Mich.) according to the manufacturer's protocol.To make a standard curve, known TNF-α concentrations were plotted andfitted to a 4 parameter logistic curve. Unknown concentrations were thenextrapolated from the standard curve.

[0151] Treatment of THP-1 cells with PDTC and compounds 1-3, 30 minutesprior to LPS activation, resulted in a significant inhibition of TNF-αproduction after a 3 hour incubation period (Table 3 and FIG. 5).Hymenialdisine 1 was found to be a potent inhibitor of, TNF-α productionwith an IC₅₀ value of 1.4 μM. Compound 2 was also found to inhibit TNF-αproduction, albeit less potent than hymenialdisine (IC₅₀=8.2 μM). Themethylated indoloazepine 3 did not have any significant activity at theconcentrations tested (tested up to 10 μM, Table 3). TABLE 3 Inhibitionof TNF-α production measured by EIA for PDTC, hymenialdisine 1 andcompounds 2 and 3 in PMA/PHA-activated THP-1 cells. Inhibition of TNF-αCompounds Production IC₅₀, (μM)^(a) PDTC (control) 6.109 (±0.529)Hymenialdisine 1 1.357 (±0.253) Compound 2 8.161 (±0.313) Compound 3  >10

EXAMPLE 5

[0152] Hymenialdisine 1 and compounds 2 and 3 were tested for theircytotoxicity and were found to exhibit significant inhibition of cellgrowth. Cells were treated at different concentrations ranging from 10μM to 5 μM concentrations over a 48 hour time period.

[0153] CEM cells (CCRF-CEM; ATCC No. CCL-119, American Type CultureCollection, Manassas, Va.) were grown in RPMI-1640 Media (Gibco-BRL,Rockville, Md.) supplemented with 10% Fetal Bovine Serum, penicillin(614 ηg/mL), streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C.,5% CO₂. DMSO was used as the vector for all drugs and added in thecontrol experiments. Cell cultures were then treated with 10 μM, 1 μM,0.1 μM or 10 μM of the drug in duplicate and allowed to incubate at 37°C., 5% CO₂. Cells were stained with 4% trypan blue solution in PBS, andcounted under the microscope (Thomas Scientific, Fisher Scientific) induplicate. This was repeated for oh, 2 h, 6 h, 12 h, 24 h, and 48 h. Thedata points obtained were then plotted and a point-to-point curve wasdrawn to determine the effect of the drug on the cells. FIG. 6A comparesthe cytotoxicity of hymenialdisine to compounds 2 and 3 and FIG. 6Bshows the growth patterns in CEM cells at different concentrations ofcompound 2. The figures show that compound 3 was less cytotoxic thancompound 2 or hymenialdisine.

[0154] Hymenialdisine and compounds 2 indicated similar inhibition ofcell growth with GI₅₀ of 1.61 μM and 1.73 μM respectively, whereascompound 3 indicated weaker inhibition of cell growth (GI₅₀=14.3 μM).The inhibition of cell growth with compounds 1-3 could therefore accountfor some of the inhibition of IL-2 production seen in the Jurkat cells.IL-2 production was measured 24 hours after PMA stimulation. However,inhibition of TNF-α production was measured within 3 hours after LPSstimulation and no significant inhibition of cell growth was apparent atthis time interval. TABLE 4 Inhibition of cell growth by hymenialdisine1 and compounds 2 and 3 in CEM leukemia cells. Inhibition of cell growthCompounds GI₅₀, (μM)^(a) PDTC (control) N/T Hymenialdisine 1 1.61(±0.062) Compound 2 1.73 (±0.088) Compound 3 14.3 (±2.41)

EXAMPLE 6

[0155] Compounds 2 and 3 were tested for inhibition of the cyclindependent kinases CDK1 and GSK-3β using the method of Meijer et al.,Chem. Biol. 7: 51-63 (2000) for showing inhibition of the kinases byhymenialdisine.

[0156] The results are shown in FIGS. 7A-C and summarized in Table 5.TABLE 5 Inhibition of CDK1 and GSK-3 by hymenialdisine 1 and compounds 2and 3. Inhibition Inhibition of CDK1 of GSK-3β Compounds IC₅₀, (μM)IC₅₀, (μM) Hymenialdisine 1 0.06 0.045 Compound 2 0.4 0.15 Compound3 >10 >10

[0157] Like hymenialdisine, compound 2 inhibited CDK1 (IC₅₀=0.4 μM) andGSK-3β (IC₅₀=150 ηM). inhibition by compound 3 was less pronounced. Theresults are consistent with the reported X-ray crystal structure ofhymenialdisine where the pyrrolic hydrogen was found to be involved in ahydrogen bonding interaction in the ATP-binding pocket (Meijer et al.,Chem. Biol. 7: 51-63 (2000)).

[0158] Table 6 illustrates the comparison between the in vitro activityof indoloazepine 2, hymenialdisine and debromohymenialdisine. Compound 2exhibits very potent inhibition of CHK2 activity in the low nanomolarrange. Interestingly, unlike the natural product hymenialdisine anddebromohymenialdisine, compound 2 exhibits also very potent selectivityfor the checkpoint kinase CHK2. TABLE 6 IC₅₀ (nM) hymenialdisine Kinasecompound 2 10, 12 debromohymenialdisine CK1δ (h) 1,352   35 NA CK2(h) >10,000 7,000 NA MEK1 (h) 89     6¹³    824¹³ PKCα (h) 2,539   700NA PKCβII (h) 3,381 1,200 NA CHK1 237 NA 3,000⁷ CHK2 8 NA 3,500⁷

EXAMPLE 7

[0159] The potent inhibitory activity of the indoloazepines againstcheckpoint kinases Chk1 and Chk2 was determined. In vitro kinase Assay:Compound 2 was tested in vitro against the kinases indicated by Upstate,UK using a Kinase Profiler Assay according to the manufacturer'sprotocol. Briefly, in a final volume of 25 μl, the kinase was incubatedwith the desired buffer and the required polypeptide substrate, inpresence of 10 mM magnesium acetate and γ-³³P-ATP (10 μM). Afterincubation for 40 minutes at room temperature, the reaction was stoppedby the addition of 3% H₃PO₄ (5 μl). A 10 μl of the reaction was thenspotted on a P30 filtermat and washed 3× in 75 mM H₃PO₄ and finally inmethanol. Samples were then dried and signals counted on a scintillationcounter. The indoloazepine exhibits very potent inhibition of Chk2activity in the low nanomolar range (IC₅₀=8 nanoMolar). Unlike thenatural product hymenialdisine and debromohymenialdisine, theindoloazepine exhibits also very potent selectivity for the checkpointkinase Chk2. These kinase inhibition studies suggest that analogs ofhymenialdisine may improve its overall kinase profile and significantlyaffect its kinase selectivity. The results are shown in FIGS. 8 and 9for CHK1 and CHK2.

[0160] While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1 1 1 22 DNA Artificial NF-kB consensus oligonucleotide 1 agttgaggggactttcccag gc 22

We claim:
 1. A compound of the formula

and acid amine salts thereof, wherein R₁, R₂′ and R₃ are moieties suchthat the compound inhibits kinases or NF-κB or NF-κB mediated geneproducts.
 2. The compound of claim 1 wherein R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide.
 3. The compound of claim 1 wherein R₁, R₂, and R₃ areeach hydrogen.
 4. The compound of claim 1 wherein R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase.
 5. Thecompound of claim 1 wherein the kinase is cyclin kinase GSK-3β or CDK-1.6. The compound of claim 1 wherein R₁, R₂, and R₃ are moieties such thatthe compound inhibits a checkpoint kinase.
 7. The compound of claim 1wherein the checkpoint kinases are Cdk1 and Cdk2.
 8. A compound of theformula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups.
 9. The compound of claim 8 wherein R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide.
 10. The compound of claim 8 wherein R₁, R₂, and R₃ areeach hydrogen.
 11. A compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups.
 12. The compound of claim 11 wherein R₁, R₂′ and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide.
 13. The compound of claim 11 wherein R₁, R₂, and R₃are each hydrogen.
 14. A compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups.
 15. The compound of claim 14 wherein R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide.
 16. The compound of claim 14 wherein R₁, R₂, and R₃are each hydrogen.
 17. A process for the preparation of a compound (IV)of the formula

wherein R₁, R₂, and R₃ are moieties which are non-reactive, whichcomprises: (a) reacting a first compound (I) of the formula

with methyl sulfonic acid and phosphorus pentoxide at elevatedtemperatures to form a second compound (II) of the formula

(b) reacting compound (II) with phenyl oxazolone and a transition metalcatalyst in a solvent to form compound (III) of the formula

and (c) reacting compound (III) with thiourea and a base in a solvent toform the compound (IV).
 18. The process of claim 17 wherein in step (b)the transition metal catalyst is titanium chloride and the solvent istetrahydrofuran.
 19. The process of claim 18 wherein in step (c) thebase is lithium hydride and the solvent is ethanol.
 20. The process ofclaim 17 wherein the compound (I) is formed by reacting a compound (V)

with an aqueous base solution.
 21. The process of claim 20 wherein thebase is lithium hydroxide.
 22. A method for inhibiting a disease in ananimal associated with a kinase, or NF-κB activation which comprises:administering a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits the kinase, NF-κB activation or NF-κBmediated gene products, the compound being administered to the animal inan amount sufficient to substantially inhibit the disease.
 23. Themethod of claim 22 wherein R₁, R₂, and R₃ are moieties such that thecompound inhibits a cyclin kinase.
 24. The method of claim 22 whereinthe cyclin kinase is GSK-3β or CDK-1.
 25. The method of claim 22 whereinR₁, R₂, and R₃ are each selected from the group consisting of hydrogen,methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl,hydroxyl, amine, thiol, ester, ether, and amide.
 26. The method of claim22 wherein R₁, R₂, and R₃ are each hydrogen.
 27. The method of claim 22wherein the disease associated with NF-κB activation is an inflammatorydisorder.
 28. The method of claim 22 wherein the disease associated withNF-κB activation is selected from the group consisting of rheumatoidarthritis, inflammatory bowel disease, asthma, dermatosis, autoimmunedisease, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, cancer, viral infections, osteoarthritis,osteoporosis, and Ataxia Telangiectasia.
 29. The method of claim 22wherein the animal is a human.
 30. A method for inhibiting production ofcytokines in an animal which comprises: administering a compound of theformula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits the cytokines, the compound beingadministered to the animal in an amount sufficient to inhibit productionof the cytokines.
 31. The method of claim 30 wherein R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase.
 32. The methodof claim 30 wherein the cyclin kinase is GSK-3β or CDK-1.
 33. The methodof claim 30 wherein R₁, R₂, and R₃ are each selected from the groupconsisting of hydrogen, methyl, alkyl containing 1 to 6 carbon atoms,halo, aryl, acyl, hydroxyl, amine, thiol, ester, ether, and amide. 34.The method of claim 30 wherein R₁, R₂, and R₃ are each hydrogen.
 35. Themethod of claim 30 wherein the cytokines are produced by activation ofthe NF-κB pathway.
 36. The method of claim 30 wherein the cytokines areselected from the group consisting of IL-2, TNF-α, and combinationsthereof.
 37. The method of claim 30 wherein the animal is a human.